Volume 74, Issue 10, Pages 1278-1286 (November 2008) Differential distribution of the Sodium-vitamin C cotransporter-1 along the proximal tubule of the mouse and human kidney  Tamara Castro, Marcela Low, Katterine Salazar, Hernán Montecinos, Manuel Cifuentes, Alejandro J. Yáñez, Juan Carlos Slebe, Carlos D. Figueroa, Karin Reinicke, María de los Angeles García, Juan Pablo Henriquez, Francisco Nualart  Kidney International  Volume 74, Issue 10, Pages 1278-1286 (November 2008) DOI: 10.1038/ki.2008.329 Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 1 Immunohistochemical detection and in situ hybridization of SVCT1 mRNA expression in the adult mouse kidney. (a) Schematic representation of adult kidney nephrons. Histological sections of the adult mouse kidney were subjected to immunohistochemistry using the specific anti-SVCT1 antisera (b, g–k) and to in situ hybridization using a specific 657bp digoxigenin-labeled riboprobe for SVCT1 mRNA (c–f). High expression of SVCT1 was observed in the proximal tubules (S3 segment) present in the outer strip of the outer medulla (c–d and g, i, and j), whereas low expression was detected in segments S2 and S1 at the inner and outer cortex (e, f and g, k). SVCT1 was specifically localized in the brush border of epithelial proximal cells (i–k, arrows). Control experiments were performed incubating the sections with antibodies pre-absorbed with an excess of the peptide used for immunization (h). C, cortex; CT, collecting tube; DCT, distal convoluted tubule; IS, inner stripe; IM, inner medulla; M, medulla; OS, outer stripe; OM, outer medulla; PCT, proximal convoluted tubule. Scale bars=100μm (b, c, and g); 20μm (d–f, h–k). Kidney International 2008 74, 1278-1286DOI: (10.1038/ki.2008.329) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 2 Brush-border membrane polarization of SVCT1 in the mouse kidney cortex and medulla. Thick floating cryosections of mouse kidney cortex (a–c) and medulla (d–f) were double stained with anti-SVCT1 (a, d, g, and h) and anti-FBPase (b, e) antibodies and secondary antibodies conjugated to Cy3 and Cy2, respectively. Merged images are shown in panels c and f. SVCT1 was detected in the apical region of epithelial cells in segments S3, S2, and S1, whereas FBPase only stained cells in segments S1 and S2. OS, outer strip; OM, outer medulla. (g–h) Pseudocolor images of SVCT1 immunostaining kidney cortex (g) and medulla (h). (i) Semiquantitative analysis plot showing relative fluorescence of SVCT1 immunocytochemical reaction in the renal cortex and medulla. Scale bars=20μm. Kidney International 2008 74, 1278-1286DOI: (10.1038/ki.2008.329) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 3 Colocalization of SVCT1 and PHA lectin binding in the mouse adult kidney. Thick floating cryosections of the mouse kidney were stained with PHA lectin (a, d, and g) and immunostained with anti-SVCT1 antibody and a secondary Cy3-conjugated antibody (b, e, and h). Merged images are shown in panels c, f, and i. Low-magnification images (a–c) show strong SVCT1 and PHA lectin staining in the outer stripe (OS) of the outer medulla (OM); however, high-magnification images show extensive colocalization of PHA lectin and SVCT1 in the brush border of proximal tubules at the cortex (d–f, arrows) and at the OS of the OM (g–i, arrows). IS/OM, inner stripe of the outer medulla. Asterisk (*), glomerulus. Scale bars=50μm (a–c); 12μm (d–i). Kidney International 2008 74, 1278-1286DOI: (10.1038/ki.2008.329) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 4 Ultrastructural immunogold labeling of SVCT1 in proximal epithelial cells of the mouse kidney. (a) Proximal tubule cells (apical region) observed with low magnification. (b) Brush-border membrane observed with high magnification. Arrows indicate gold particles on the microvilli. Original magnification were (a) × 17,000; (b) × 35,000. Kidney International 2008 74, 1278-1286DOI: (10.1038/ki.2008.329) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 5 Immunohistochemical detection of SVCT1 expression in the adult human kidney. Histological sections of the adult human kidney were subjected to immunohistochemistry using the specific anti-SVCT1 antisera and a peroxidase-conjugated secondary antibody. (a) A low-magnification image shows intense immunostaining for SVCT1 in the proximal tubules situated in the medullary ray (MR) and cortex. High-magnification images show that SVCT1 immunostaining is strongly concentrated at the brush-border membrane of epithelial cells of the proximal convoluted (b–d) and straight (e) tubules and MRs (d, e). Low-to-absent expression was detected in the cells of the distal convoluted tubes (DCT). PCT, proximal convoluted tubule; PST, proximal straight tubule; G, glomerulus. Scale bars=20μm (a–e). Kidney International 2008 74, 1278-1286DOI: (10.1038/ki.2008.329) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 6 RT-PCR, western blot, and immunocytochemistry for SVCT1 in LLC-PK1 cells. (a) mRNAs isolated from adult human (lane 1) and mouse (lane 2) kidneys as well as from LLC-PK1 cells (lane 3) were subjected to RT-PCR with specific primers against SVCT1 transporter. Lane 4, reverse transcriptase-negative reaction for LLC-PK1 cells mRNA. As controls, RT-PCRs were performed with specific primers against β-actin (lower panel). Arrows in the right of each panel indicate the expected RT-PCR product size for mouse SVCT1 (439bp) and β-actin (333bp). (b) Western blot analysis using specific anti-SVCT1 antibodies detected a 68kDa band in membrane protein extracts from the mouse kidney and LLC-PK1 cells (lanes 1 and 2, respectively). As controls, western blots were developed avoiding primary antibodies (lane 3). (c) Immunocytochemical analysis of SVCT1 (green) in pre-confluent (c) and post-confluent (d) LLC-PK1 cells. Cell nuclei in panel c were stained with propidium iodide. Inset in panel c shows negative controls and primary antibodies were avoided. Lower (x–z) panel in panel d show confocal z-sections of the plane indicated with white line in panel d. Scale bar=20μm. Kidney International 2008 74, 1278-1286DOI: (10.1038/ki.2008.329) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 7 Kinetic analysis of L-ascorbate uptake in LLC-PK1 cells. (a) Time course of 50μM AA uptake at 37°C in the presence of NaCl (open circles) or replacing NaCl with choline chloride (solid circles). (b) Uptake at 10min of 50μM AA in LLC-PK1 cells that were treated with choline chloride, 10μM ouabain for 1h, and 25μM cytochalasin B for 10min. Results are expressed as percentage of the control (*P<0.001). (c) Dose-response curve of L-ascorbic acid at 37°C. (d) Eadie–Hofstee transformation of the data in panel c. (e) Velocity of 50μM AA uptake at 10min as a function of the concentration of extracellular sodium at 37°C. (f) Hill plot of the data in panel e. (g) Uptake of 50μM AA at 5min between 4 and 37°C. (h) pH dependence of 50μM AA uptake at 5min. Data represent the mean±s.d. of at least two experiments performed in triplicate. Kidney International 2008 74, 1278-1286DOI: (10.1038/ki.2008.329) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 8 SVCT1 detection and function in LLC-PK1 cells treated with different ascorbic acid concentration. (a) Immunocytochemical analysis of SVCT1 in post-confluent LLC-PK1 cells incubated with different concentrations of AA (C, 10, 25, 50, and 100μM). Lower panel (x–z) show confocal z-sections of the planes indicated with white lines. (b) Uptake at 10min of 100μM AA in LLC-PK1 cells that were treated with different concentrations of the vitamin. As a control (c), we used LLC-PK1 cells cultured with a low AA concentration (probably lesser than 10μM) present in the serum added to the culture medium. Data represent the mean±s.d. of at least two experiments performed in triplicate. Scale bar=10μm (a). Kidney International 2008 74, 1278-1286DOI: (10.1038/ki.2008.329) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 9 L-Ascorbic acid uptake in LLC-PK1 cells pretreated and incubated with different concentrations of vitamin C. LLC-PK1 cells were cultured with 10, 25, 50, and 100μM AA and the vitamin C uptake was performed using the same concentration in each point. As a control (C), we incubated LLC-PK1 cells with a low AA concentration (probably lesser than 10μM) present in the serum added to the culture medium. Data represent the mean±s.d. of at least two experiments performed in triplicate. Kidney International 2008 74, 1278-1286DOI: (10.1038/ki.2008.329) Copyright © 2008 International Society of Nephrology Terms and Conditions